Use of N-acyl derivatives of aminoalcohols in the manufacture of a medicament for practicing neuroprotective action in neuropathological states connected with excitotoxicity

ABSTRACT

The use of N-acyl derivatives of aminoalcohols with mono- and di-carboxylic acids for the prevention and treatment of diseases connected with hyper and prolonged excitation by excitatory amino acids is described.

FIELD OF THE INVENTION

The present invention relates to the use of N-acyl derivatives ofaminoalcohols with mono and dicarboxylic acids for the prevention andtreatment of neuropathological states connected with excitotoxicity.

PRIOR ART DISCLOSURE

Recent research has established that the nervous system is not anisolated entity but a component of a complex intercommunication systemtogether with the main homeostatic systems--immune system and endocrinesystem--where stimuli from one of the three systems are received andadaptive responses are processed by the other two in order to maintainor regulate homeostatic equilibrium in the body. In fact, although thenervous system, the endocrine system and the immune system utilizelanguages of their own, they can translate and process the messagesreceived from the other systems.

Scientific literature has recently reported some results suggesting thatthe mast cell, an immunocompetent cell ubiquitously present in tissues,might be the element providing communication between the three systems:due to the anatomic dislocation--in proximity of nerve endings and incontiguity with the vascular system--and to its functional role beingevidenced, said cell might be capable of acting as a "gate keeper"within said complex system.

Following tissue injury, stimuli capable of activating mast cells areproduced leading to the prompt release from preformed stores ofmediators and other substances with chemotactic, proinflammatory andcytotoxic effects. Cytokines (TNF-α and interleukins), vasoactive amines(serotonin, bradikinin and histamine), heparin and PAF are someexamples.

The presence of mediators and kinins at the site of injury increases thesensitivity to pain in the acute stage; further, the accumulation ofIL-1, IL-3 and TNF-α induces the synthesis of NGF which locallyincreases the proinflammatory agonist stimulus (mast cell andT-lymphocyte proliferation, mast cell degranulation), which is thusamplified and prolonged (U. Otten et al., 1989, "Nerve Growth Factorinduces growth and differentiation of human lymphocytes", PNAS 86:10059-10063; J. S. Marshall et al., 1989, "The role of mast celldegranulation product in mast cell hyperplasia", The J. of Immunology,144: 1886-1892) having the consequence of inducing modifications in thenumber of nerve endings and facilitate these inflammatory conditions tobecome chronic (R. Dubner and M. A. Ruda, 1992, "Activity-dependentneuronal plasticity following tissue injury and inflammation", TINS, 15,3: 96-103).

These and further evidences for the susceptibility and rapidity of mastcell degranulation phenomena suggested the need for an inhibitorycontrol. In addition to the corticosteroid-mediated pleiotropic control,already characterized, a local control system has been developed by theApplicant, said system being pharmacologically applicable with specificcompounds structurally related to endogenous autacoids and chemicallydefinable as N-acyl lipids, as disclosed in European patent applicationsNo. 92121862.4 published with No. 0 550 006 A3 and No. 92121864.0published with No. 0 550 008 A2.

The experimental evidence obtained by the Applicant for these N-acyllipids proved the presence of a local antagonist endogenous regulationmechanism, meant to control mast cell degranulation induced byneurogenic and immunogenic supramaximal stimuli (ALIA=Autacoid LocalInflammation Antagonism). Said discovery is of great pharmacologicalimportance, making it possible to synthesize drugs, chemically definableas N-acyl derivatives of mono and polycarboxylic acid withaminoalcohols, which can act as autacoids with the ability to modulatemast cell activation associated to neuroimmunogenic inflammatoryprocesses in autoimmune diseases and other pathological conditions (seethe aforementioned patent applications).

SUMMARY OF THE INVENTION

The Applicant has surprisingly found that compounds belonging to theclass of N-acyl derivatives of mono and dicarboxylic acids withaminoalcohols which can exert a protective action against theneurotoxicity induced by excitatory amino acids (EAA), such as glutamicacid (hereinafter referred to as Glu), in neuronal cultures, whenincubated at the same time or after exposure to the excitotoxicstimulus. The kinetics of this effect makes said compounds of highapplicative interest in acute and chronic disorders of the centralnervous system. Therefore, the present invention is related to the useof said N-acyl derivatives and of more soluble and/or slow releasederivatives thereof as active principles in the preparation ofpharmaceutical compositions for the prevention and treatment of acuteand chronic diseases of the central nervous system connected with EAAneurotoxicity.

The compounds according to the present invention are defined by thefollowing formulas (I) and (II): ##STR1## where ##STR2## is the acylradical of a linear or branched, saturated or unsaturated aliphaticmonocarboxylic acid, containing 2 to 20 carbon atoms, optionallysubstituted in the aliphatic chain with one of more hydroxyl, aminic,ketonic, carboxyl, cycloalkyl, aryl, heterocyclic, aromatic,heteroaromatic, polycyclic groups; or the acyl radical of an aromatic,heteroaromatic or heterocyclic monocarboxylic acid; ##STR3## is thediacyl radical of a saturated or unsaturated aliphatic dicarboxylicacid, optionally substituted with an aminic, hydroxyl or ketonic group;or the diacyl radical of an aromatic, heterocyclic or heteroaromaticdicarboxylic acid;

R₂ and R₅ are alcoholic residues selected from a C₁ -C₂₀ linear orbranched hydroxyalkyl, optionally substituted in the aliphatic chainwith one or more aryl groups, and a hydroxyaryl optionally substitutedwith one or more linear or branched alkyl radicals of from 1 to 20carbon atoms; in both cases, the hydroxyl group is optionally esterifiedto --OX, wherein X is the acyl radical of a linear or branched,saturated or unsaturated, aliphatic acid, optionally substituted in thealiphatic chain with one or more aryl groups or with one --COOHoptionally salified, or X is the acyl radical of an aromatic acid or--PO₃ H₂ optionally salified;

R₃ is H or R₂ ;

R₆ is H or R₅.

DETAILED DESCRIPTION OF THE INVENTION

The characterization and advantages of N-acyl derivatives of mono anddicarboxylic acids of aminoalcohols as active compounds able tocounteract the neuronal death induced by excitotoxic event and,therefore, useful for the treatment of diseases connected with a hyperand prolonged stimulation of EAA receptors will be described in detailhereinafter.

In the derivatives of formula (I),

when ##STR4## the acyl radical of a saturated or unsaturated aliphaticmonocarboxylic acid, it is preferably selected from the group consistingof acetic, caproic, butyric, palmitic, oleic, stearic, lauric, linoleic,linolenic and myristic acid and hydroxyl, aminic and ketonic homologuesthereof, glycolic, pyruvic, lactic, caprylic, valeric, valproic,arachidonic acid, gamma-trimethyl-β-hydroxybutyrobetaine and derivativesthereof acylated on the hydroxyl group;

when ##STR5## is the acyl radical of an aromatic, heteroaromatic orheterocyclic monocarboxylic acid, it is preferably selected from thegroup consisting of salicylic, acetylsalicylic, benzoic,trimethoxybenzoic, isonicotinic, thenoic, phenylanthranilic, retinoic,hydroxyphenylaceric, α-lipoic (thioctic) and deoxycholic acid.

In the derivatives of formula (II),

when ##STR6## is the diacyl radical of a saturated or unsaturatedaliphatic dicarboxylic acid, it is preferably selected from the groupconsisting of oxalic, fumaric, azelaic, succinic, traumatic, glutaricand muconic acid and hydroxyl, aminic and ketonic homologs thereof,malic, aspartic and tartaric acid;

when ##STR7## is the diacyl radical of an aromatic, heteroaromatic orheterocyclic dicarboxylic acid, it is preferably selected from the groupconsisting of phthalic, folic and chromoglycic acid.

R₂, R₃, R₅, and R₆, are preferably radicals of aminoalcohols selectedfrom the group consisting of monoethanolamine, diethanolamine,2-hydroxypropylamine and di-(2-hydroxypropyl)-amine, wherein thehydroxyl group is optionally esterified to aliphatic esters, araliphaticesters, aromatic esters, O-phosphates, acid hemiesters or salifiedderivatives thereof.

Therefore, with reference to formula (I), the compounds of the presentinvention have preferably the following structures: ##STR8## where X isH, --PO₃ H₂, optionally salified with monovalent or bivalent inorganicions, or X is the acyl radical of a linear or branched, saturated orunsaturated, aliphatic carboxylic acid, optionally substituted on thealiphatic chain with one or more aryl groups or with one --COOHoptionally salified with monovalent or bivalent inorganic ions, and ispreferably ##STR9## or X is the acyl radical of an aromatic carboxylicacid, preferably benzoic acid.

Said monovalent or bivalent inorganic ions are preferably K, Na, Mg orCa.

With reference to formula (II), the compounds of the present inventionhave preferably the following structures: ##STR10## where X is definedas above.

It is to be noted that chemical group X serves to increase thesolubility in water and/or to modify the pharmacokinetic properties ofthe compound, giving a pro-drug.

It is also to be noted that the derivatives of the aminoalcohols2-hydroxypropylamine and di-(2-hydroxypropyl)-amine can be racemic oroptical isomers.

The compounds according to the present invention are prepared by thedifferent methods described hereinbelow.

Method of Synthesis A

High-temperature melting of alkanolamine salt with a carboxylic acid,with formation of the relevant alkanolamide.

Procedure

A mixture consisting of a carboxylic acid and an alkanolamine (1:1.5equivalents) is fed to a flask provided with reflux condenser and heatedto 130-160° C., for 4 to 8 hrs, on an oil bath. The alkanolamideobtained is purified by a method or by a combination of methods reportedbelow:

fractional distillation in vacuo;

liquid/liquid extraction;

crystallization from a suitable solvent or mixture of volatile solvents,selected from the classes of alcohols, ketones, esters, ethers,hydrocarbons or chlorinated solvents;

liquid chromatography using an ion exchange resin, silica gel or aluminaas stationary phase, or a reversed phase adsorbent selected among thoseavailable on the market, consisting of polymers or silica.

Method of Synthesis B

N-acylation of an alkanolamine with a suitable carboxylic acid activatedderivative.

Procedure

An alkanolamine solution in a suitable solvent or mixture of solvents,selected from the group consisting of water, alcohols, ketones, esters,ethers, chlorinated solvents, dialkylamides, dialkylsulphoxides andheterocycles, is cold stirred. The carboxylic acid activated derivative(acyl halide, anhydride or alkylester) is added slowly over a period of30 min to 3 hrs.

Acylation can be carried out in the presence of a suitable base,selected from the group consiting of hydroxides, inorganic carbonatesand bicarbonates and organic tertiary amines, at a temperature rangingfrom -20° C. to +120° C. The alkanolamide obtained is purified by amethod or by a combination of methods as per Method A.

Method of Synthesis C

Activation of the carboxylic acid with alkylchloroformate, followed byaminolysis of the obtained mixed anhydride with alkanolamine.

Procedure

A carboxylic acid solution in a suitable organic solvent, selected fromthe group consisting of ketones, esters, ethers, chlorinated solvents,dialkylamides, dialkylsulphoxides and heterocycles, is cold stirred at atemperature ranging from -20° C. to +30° C. An equivalent quantity ofalkylchloroformate is added slowly, over a period of 30 min to 3 hrs.

Activation is carried out in the presence of a suitable weak base,selected from the group consisting of inorganic carbonates andbicarbonates and organic tertiary amines, at a temperature ranging from-20° C. to +30° C.

The obtained mixed anhydride is subjected to aminolysis by addition ofan equivalent quantity of alkanolamine, still at low temperature.

The alkanolamide obtained is purified by a method or by a combination ofmethods as per Method A.

Some preparation examples of the compounds of formulas (I) and (II)according to the present invention and their biological activities arereported hereinbelow for illustrative but not limitative purposes.

EXAMPLE 1 Preparation of N-(2-Hydroxyethyl)-Palmitoylamide

Following the method described by Roe et al. (J.Am.Chem.Soc., 74,3442-3443, 1952), palmitic acid (2.56 g; 1 mmol) was caused to reactwith ethanolamine (0.9 g; 1.5 mmol) by refluxing in ethyl ether, undernitrogen atmosphere, for 5 to 6 hrs.

The product obtained was extracted from the reaction mixture andcrystallized from 95% ethyl alcohol at 0° C.

The physico-chemical properties of N-(2-hydroxyethyl)-palmitoylamidewere as follows:

    ______________________________________    physical state               crystalline powder    molecular formula               C.sub.18 H.sub.37 NO.sub.2    molecular weight               299.48    elemental analysis               C = 72.19%; H = 12.45%; N = 4.68%; O = 10.69%    solubility in organic               hot methanol, CH.sub.3 Cl, DMSO    solvents    solubility in water               insoluble    melting point               94-95° C.    TLC        eluent: chloroform-methanol, 9/1               Rf = 0.75    ______________________________________

EXAMPLE 2 Preparation of N-(2-Acetoxyethyl)-Palmitoylamide

N-(2-hydroxyethyl)-palmitoylamide (see Example 1) (3.0 g; 10 mmol) wassolubilized in anhydrous pyridine (30 ml) under stirring at 0° C. andadded with acetic anhydride (1.53 g; 15 mmol). The resulting solutionwas stirred at 0° C. for a period of 15 min and heated to 45° C. for 24hrs. The reaction mixture was evaporated to dryness in vacuo. Theresidue was taken up with methanol (30 ml) and evaporated to dryness invacuo. The residue was crystallized from methanol (50 ml) and thecrystalline fraction was separated by filtration, washed three timeswith cold methanol (10×3 ml) and finally dried in high vacuo.

The reaction yield was 93% approx.

The physico-chemical properties of N-(2-acetoxyethyl)-palmitoylamidewere as follows:

    ______________________________________    physical state               white crystalline powder    molecular formula               C.sub.20 H.sub.39 NO.sub.3    molecular weight               341.54    elemental analysis               C = 70.34%; H = 11.51%; N = 4.10%; O = 14.05%    solubility in organic               >5 mg/ml in n-octanol    solvents    solubility in water               poorly soluble    melting point               78-81° C.    TLC        eluent: chloroform-methanol, 95/5               Rf = 0.58    ______________________________________

EXAMPLE 3 Preparation of N-(2-Acetoxyethyl)-Benzoylamide

N-(2-hydroxyethyl)-benzoylamide (1.65 g; 10 mmol), prepared according toExample 8 of the European patent application published with No. 0 550006, was solubilized in anhydrous pyridine (15 ml) under stirring at 0°C. and added with acetic anhydride (1.53 g). The resulting solution wasstirred at 0° C. for a period of 15 min and heated to 45° C. for 24 hrs.The reaction mixture was evaporated to dryness in vacuo. The residue wastaken up with water (30 ml) and extracted twice with ethyl acetate (20×2ml). The organic phases were washed twice with 1 N HCl (10×2 ml), oncewith water (10 ml), twice with 5% NaCOH₃ (10×2 ml) and once with water(10 ml). They were then combined, dehydrated with Na₂ SO₄, evaporated todryness in vacuo. The residue was crystallized from tert-butylmethylether (20 ml) and the crystalline fraction was separated by filtration,washed twice with cold tert-butylmethyl ether (5×2 ml) and finally driedin high vacuo.

The reaction yield was 85% approx.

The physico-chemical properties of N-(2-acetoxyethyl)-benzoylamide wereas follows:

    ______________________________________    physical state               white crystalline powder    molecular formula               C.sub.11 H.sub.13 NO.sub.3    molecular weight               207.23    elemental analysis               C = 63.76%; H = 6.32%; N = 6.76%; O = 23.16%    solubility in organic               >10 mg/ml in n-octanol;    solvents   >10 mg/ml in DMSO    solubility in water               poorly soluble    melting point               52.5-54.5° C.    TLC        eluent: chloroform-methanol-water-NH.sub.3               (30%), 80/25/2/1               Rf = 0.77    ______________________________________

EXAMPLE 4 Preparation of N,N'-Bis(2-Acetoxyethyl)-Fumaroylamide

N,N'-bis(2-hydroxyethyl)-fumaroyldiamide (2.02 g; 10 mmol), preparedaccording to Example 1 of the European patent application published withNo. 0 550 008, was solubilized in anhydrous pyridine (20 ml) understirring at 0° C. and added with acetic anhydride (3.06 g; 30 mmol). Theresulting solution was stirred at 0° C. for a period of 15 min andheated to 45° C. for 24 hrs. The reaction mixture was evaporated todryness in vacuo. The residue was taken up with methanol (30 ml) andevaporated to dryness in vacuo. The residue was crystallized fromabsolute ethanol (50 ml) and the crystalline fraction was separated byfiltration, washed three times with cold ethanol (5×3 ml) and finallydried in high vacuo.

The reaction yield was 91% approx.

The physico-chemical properties ofN,N'-bis(2-acetoxyethyl)-fumaroylamide were as follows:

    ______________________________________    physical state               white crystalline powder    molecular formula               C.sub.12 H.sub.18 N.sub.2 O.sub.6    molecular weight               286.29    elemental analysis               C = 50.35%; H = 6.33%; N = 9.79%; O = 33.53%    solubility in organic               >10 mg/ml in DMSO    solvents    solubility in water               poorly soluble    melting point               214-216° C.    TLC        eluent: chloroform-methanol-water-NH.sub.3               (30%), 80/25/2/1               Rf = 0.81    ______________________________________

EXAMPLE 5 Preparation of N-Palmitoylethanolamide Phosphate

N-(2-hydroxyethyl)-palmitoylamide (see Example 1) (3.0 g; 10 mmol) wassolubilized in anhydrous methanesulphonic acid (10 ml) under stirring at0° C. and added with phosphoric anhydride (2.12 g; 15 mmol). Theresulting mixture was stirred at 0° C. for a period of 25 hrs. Thereaction mixture was added with ether until product precipitation wascomplete. The precipitate was separated by centrifugation, dried invacuo, washed with cold water and finally dried in vacuo. The crudeproduct obtained was hot washed with tert-butylmethyl ether (50 ml) andcrystallized from isopropanol (50 ml). The crystalline fraction wasseparated by filtration, washed three times with cold isopropanol (10×3ml) and finally dried in high vacuo.

The reaction yield was 83% approx.

The physico-chemical properties of N-palmitoylethanolamide phosphatewere as follows:

    ______________________________________    physical state               white crystalline powder    molecular formula               C.sub.18 H.sub.38 NO.sub.5 P    molecular weight               379.48    elemental analysis               C = 56.97%; H = 10.09%; N = 3.69%; O = 21.08%;               P = 8.16%    solubility in organic               >10 mg/ml in DMSO    solvents    solubility in water               poorly soluble (>1 mg/ml in 50 mM               phosphate buffer, pH 7.4, NaCl 0.9%)    melting point               undeterminable    TLC        eluent: chloroform-methanol-water-NH.sub.3               (30%), 50/40/7/3               Rf = 0.38    ______________________________________

EXAMPLE 6 Preparation of N,N'-Bis(2-Hydroxyethyl)-DodecenediamideDiphosphate Disodium Salt

Traumatic acid (2.28 g; 10 mmol) and N-hydroxysuccinimide (2.42 g; 21mmol) were solubilized in anhydrous pyridine (50 ml) under stirring at0° C. and added with dicyclohexylcarbodiimide (4.33 g; 21 mmol). Theresulting mixture was stirred at 0° C. for a period of 1 hr and furtherstirred at room temperature for a period of 20 hrs. The resultingsuspension was filtered, the precipitate removed and the solutionevaporated to dryness in vacuo. The crude residue was solubilized in DMF(50 ml), added with triethylamine (5.05 g; 50 mmol) and stirred at 0° C.O-phosphocholamine (2.96 g; 21 mmol) was solubilized in cold water (10ml) and the resulting solution was added dropwise to the succinimideester solution over a period of 1 hr. The mixture was further stirred atroom temperature for a period of 24 hrs and finally evaporated todryness in high vacuo. The residue was taken up with water (20 ml) andeluted in a column containing 70 ml of cationic exchange resin Dowex50×8, generated in the H+ form. The eluate was neutralized to pH 7.0with 5% Na₂ CO₃ solution and concentrated in vacuo to 5 ml by vol.approx. The oily residue was purified by reverse phase chromatography inLichrosorb RP18 R column, eluting with water. The fractions containingthe pure product were combined and lyophilized.

The reaction yield was 78% approx.

The physico-chemical properties ofN,N'-bis-(2-hydroxyethyl)-dodecenediamide diphosphate disodium salt wereas follows:

    ______________________________________    physical state               white amorphous powder    molecular formula               C.sub.16 H.sub.30 N.sub.2 O.sub.10 P.sub.2 Na.sub.2    molecular weight               518.35    elemental analysis               C = 37.07%; H = 5.83%; N = 5.40%; O = 30.87%;               P = 11.95%; Na = 8.87%    solubility in organic               >10 mg/ml in DMSO (product in acid form)    solvents    solubility in water               >10 mg/ml    melting point               214-216° C.    TLC        eluent: ethanol/water/acetic acid,               70/20/10               Rf = 0.47    ______________________________________

EXAMPLE 7 Preparation of N-(2-Hydroxyethyl)-Palmitoylamide SuccinateAcid

N-(2-hydroxyethyl)-palmitoylamide (3.0 g; 10 mmol) (see Example 1) wasadded with succinic anhydride (1.5 g; 15 mmol) and anhydrous sodiumacetate (50 mg). The mixture was heated to 120° C. with continuedstirring, under nitrogen atmosphere, and cooled to room temperature. Thecrude product obtained was crystallized from a 0:30 ethanol-watermixture (50 ml). The precipitate was separated by filtration, washedthree times with cold ethanol (10×3 ml) and finally dried in high vacuo.

The reaction yield was 94% approx.

The physico-chemical properties of N-(2-hydroxyethyl)-palmitoylamidesuccinate acid were as follows:

    ______________________________________    physical state               white crystalline powder    molecular formula               C.sub.22 H.sub.41 NO.sub.5    molecular weight               399.58    elemental analysis               C = 66.13%; H = 10.34%; N = 3.51%; O = 20.02%    solubility in organic               >5 mg/ml in DMSO    solvents    solubility in water               poorly soluble (>1 mg/ml as potassium               salt)    melting point               115-118° C.    TLC        eluent: chloroform-methanol-water-NH.sub.3               (30%), 80/25/2/1               Rf = 0.22    ______________________________________

EXAMPLE 8 Preparation of N,N'-Bis(2-Hydroxyethyl)-DodecenediamideDisuccinate Acid

N,N'-bis(2-hydroxyethyl)-dodecenediamide (3.14 g; 10 mmol) preparedaccording to Example 6 of the European patent application published withNo. 0 550 008, was added with succinic anhydride (3.0 g; 30 mmol) andanhydrous sodium acetate (100 mg). The mixture was heated to 120° C. for3 hrs with continued stirring, under nitrogen atmosphere, and cooled toroom temperature. The crude product obtained was crystallized from a70:30 ethanol-water mixture (50 ml). The precipitate was separated byfiltration, washed three times with cold ethanol (10×3 ml), and dried inhigh vacuo.

The reaction yield was 89% approx.

The physico-chemical properties ofN,N'-bis(2-hydroxyethyl)-dodecenediamide disuccinate acid were asfollows:

    ______________________________________    physical state               white amorphous powder    molecular formula               C.sub.24 H.sub.38 N.sub.2 O.sub.10    molecular weight               514.58    elemental analysis               C = 56.02%; H = 7.44%; N = 5.44%; O = 31.09%    solubility in organic               >10 mg/ml in DMSO    solvents    solubility in water               poorly soluble (>1 mg/ml in 50 mM               phosphate buffer, pH 7.4, NaCl 0.9%)    TLC        eluent: chloroform-methanol-water-NH.sub.3               (30%), 50/40/7/3               Rf = 0.56    ______________________________________

EXAMPLE 9 Preparation of N,N'-Bis-(2-Hydroxyethyl)-Nonandiamide

A mixture of acrylic acid (1.88 g; 10 mmol) and ethanolamine (1.84 g; 30mmol) was fed to a flask provided with reflux condenser and heated to160° C., for 6 hrs, on an oil bath.

The reaction mixture was crystallized from isopropanol (50 ml). Thecrystalline fraction was separated by filtration, washed three timeswith cold isopropanol and finally dried in high vacuo.

The reaction yield was 78% approx.

The physico-chemical properties of N,N'-bis(2-hydroxyethyl)-nonandiamidewere as follows:

    ______________________________________    physical state               white crystalline powder    molecular formula               C.sub.13 H.sub.26 N.sub.2 O.sub.4    molecular weight               274.37    elemental analysis               C = 56.91%; H = 9.55%; N = 9.55%; O = 23.3%    solubility in organic               >10 mg/ml in DMSO    solvents    melting point               132-134° C.    TLC        eluent: chloroform-methanol-water-NH.sub.3               (28%), 80/25/2/1               Rf = 0.48    ______________________________________

EXAMPLE 10 Preparation of N-(2-Hydroxyethyl)-Arachidonamide

A mixture of arachidonic acid (3.04 g; 10 mmol) and triethanolamine(10.6 g; 10.5 mmol) in anhydrous THF (100 ml) was stirred under nitrogenatmosphere, added dropwise over a period of 30 minutes to a solution ofisobutylchloroformate (1.44 g; 10.5 mmol) in THF (50 ml). The resultingmixture was stirred at -10° C. for 2 hrs and then at 0° C. for 5 hrs,and added dropwise with ethanolamine (0.9 g; 14.7 mmol). The resultingmixture was stirred at 0° C. for over 2 hrs and finally evaporated todryness. The crude residue was purified by chromatography eluting with a98:2 v/v chloroform-methanol mixture. The eluate fractions were combinedand evaporated to dryness; the residue was dried in vacuo.

The physico-chemical properties of N(2-hydroxyethyl)-arachidonamide wereas follows:

    ______________________________________    physical state               colourless oil    molecular formula               C.sub.22 H.sub.37 NO.sub.2    molecular weight               347.54    elemental analysis               C = 76.03%; H = 10.73%; N = 4.03%; O = 9.21%    solubility in organic               >10 mg/ml in DMSO; >10 mg/ml in ethanol    solvents    solubility in water               poorly soluble    TLC        eluent: chloroform-methanol-water-NH.sub.3               (28%), 80/25/2/1               Rf = 0.66    ______________________________________

BIOLOGICAL ACTIVITY

In vitro evaluation of the neuroprotective effect against excitotoxicityof Excitatory Amino Acids in primary neuronal cultures of the followingcompounds was carried out:

Compound a: N-(2-hydroxyethyl)-palmitoylamide (Example 1)

Compound b: N-palmitoylethanolamide phosphate (Example 5)

Compound c: N-(2-hydroxyethyl)-arachidonamide (Example 10)

Compound d: N,N'-bis-(2-hydroxyethyl)-nonanediamide (Example 9)

Materials and Methods

Compounds Solubilization

The compounds under evaluation, referred to as a, b, c, and d, weresolubilized in DMSO and culture medium to obtain concentrations of 100μM (4% DMSO) and 60 μM (3% DMSO) and added to the culture mediumsaccording to different treatment schedules in order to determine thetime-effect relationship vs. the incubation time and the exposure to theneurotoxic stimulus.

Cultures Preparation

Granular cell cultures were obtained from postnatal day 8-9 mouse Balb-6cerebellum. The cells were suspended in EBM+2 mM L-glutamine, 100 U/mlpenicillin, 50 μg/l gentamicin, 25 mM KCl and 10% fetal calf serum,plated on polylysine substrate, cultured for 8-10 days and then exposedto Glu 500 μM at room temperature for 5 min. The compounds beingevaluated were added to the cultures at the same time of exposure, soonafter exposure or 15 min after exposure to Glu, and cultured for 60minutes; cultures were then washed and brought back to the originalmedium. The number of survived cells was evaluated by calorimetricmethod (MTT) 24 hours after excitotoxic stimulation.

Results

The tested compounds exert a protective action against the excitotoxicstimulus at the two concentrations tested (100 and 60 μM), independentlyof the treatment scheme adopted, that is when incubated at the same timeof exposure, soon after exposure or 15 min after exposure to Glu, asshown in Table 1. Furthermore, they do not show any significantintrinsic cytotoxicity.

Said experimental evidences suggest that the aforesaid molecules exert aprotective action within very short times and affect not only thecytotoxic events mediated by an overstimulation of NMDA receptor, butabove all, being active during post-treatment, the cytotoxic mechanismsfollowing receptor activation, thus concurring for example to limitcalcium ions influx into the cell and the consequent processes leadingto cellular death.

                  TABLE 1    ______________________________________    Protective effect against cellular death induced by Glu (500 μM    for 5 min) of compound a, b, c and d, incubated at the concentrations    of 60 and/or 100 μM and added to the cells:    A: at the same time of exposute to Glu;    B: soon after exposure to Glu;    C: 15 min after exposure to Glu.    Cell survival is expressed as a percentage of control values. Each value    is the average of three tests. Incubation with 4% DMSO does not affect    cell survival.                   survived cells (%)    Treatment        100 μM                             60 μM    ______________________________________    Control values   100     100    Glu              40      44    A:    + Compound a     67      86    + Compound b     n.d.    68    + Compound c     n.d.    95    + Compound d     68      n.d.    B:    + Compound a     84      n.d.    + Compound b     n.d.    99    + Compound d     92      n.d.    C:    + Compound a     76      101    + Compound b     n.d.    90    + Compound d     87      n.d.    ______________________________________

Conclusions

As a whole, the above experimental evidences show that N-acylderivatives of mono and dicarboxylic acids with amino alcohols accordingto the present invention are able, even at low concentrations, to exerta specific protective action against the neurotoxicity of ExcitatoryAmino Acids on neuronal cells. Particular notice has to be given to theoutstanding therapeutic significance of said molecules, in that i) theyare characterized by an extremely prompt effect and ii) they are activewhen administered at the same time or more importantly after exposure toexcitatory amino acids, thus affecting not only the short-termneurotoxic mechanisms mediated by the activation of glutamic or kainicacid receptors, but also one or more events downstream of receptoroverstimulation. Furthermore, the compounds do not show any significantintrinsic cytotoxicity.

Therefore, the derivatives described herein can be advantageously usedin the treatment of CNS disorders of humans and animals, whoseetiology/evolution is associated with the hyper stimulation ofexcitatory amino acid receptors, in particular the NMDA receptor.

In fact, under physiological conditions, Excitatory Amino Acids (EAA)mediate synaptic transmission, involved in the phenomena of neuronalplasticity underlying behavioural and cognitive processes, and in motorfunctions. Under particular conditions of hyper stimulation, they areresponsible for a neuronal damage that may ultimately result in neuronaldeath. There is evidence for a correlation between overstimulation ofNMDA receptors and CNS damage associated with acute events, such ashypoxia-ischemia, stroke, hypoglycemia, perinatal anoxia, epilepsy,brain and spinal cord injuries, neurolathyrism, or in chronicneurodegenerative disorders, such as Hungtington's Chorea, Alzheimer'sand Parkinson's diseases, amyotrophic lateral sclerosis, andpontocerebellar degeneration (J. W. Olney, "Excitotoxic Amino Acids andNeuropsychiatric Disorders", Ann.Rev.Pharmacol.Toxicol., 30: 47-71,1990) or neurological complications associated with viral diseases, suchas HIV-1 infection (AIDS dementia complex) (M. P. Heyes et al.,"Quinolinic Acid in Cerebrospinal fluid and serum in HIV-1 Infection:Relationship to Clinical and Neurological Status", Ann.Neurol., 29:202-209, 1991).

Further disturbances of the glutamatergic system are known to have aremarkable consequence for hypothalamic homeostasis and on processesderived therefrom (A. N. Van den Pol and P. Q. Trombley, "GlutamateNeurons in Hypothalamus regulate Excitatory Transmission", The J. ofNeuroscience, 13(7): 2829-2836, 1993).

It should be emphasized that currently available therapies are oflimited value in the acute phase of diseases such as cerebral ictus,which have an extremely narrow "therapeutic window" in terms of the timeframe during which neuronal cell death can be limited by pharmacologicalintervention. In this context, it is of great significance that themolecules of the present invention are capable of acting not only withinthis very short time of damage induction, but also later on when thedamage has been already established. Further, since said new derivativesdo not act like NMDA-receptor competitive or non-competitive inhibitors,they may effectively be used also to treat chronic diseases such asHungtington's Chorea, Parkinson's disease and conditions involvingdementia without affecting plastic phenomena and thus, unlike NMDAreceptor inhibitors, would not be expected to worsen the clinicaloutcome.

It needs to be remembered that dosages, times and routes ofadministration will vary depending on the disease type, stage andseverity. A distinction is to be made between treatment of:

i) acute conditions (hypoxia-ischemia, stroke, brain and spinal cordinjury, hypoglycemia, cerebral hypoxic states associated withcardiovascular surgery) or diseases inducing a prompt and massive Glurelease, such as epilepsy, transient ischemic attacks (TIA),neurolathyrism;

ii) chronic degenerative diseases such as Hungtington's Chorea,Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis anddementia, either primary or associated with other pathologies even ofviral origin, such as acquired immunodeficiency syndrome (AIDS dementiacomplex).

Further, primary retinic diseases even of anoxic nature or associatedwith eye hypertension, e.g. glaucoma, are to be taken intoconsideration. For all aforesaid diseases, the compounds according tothe present invention can be administered by systemic oral or parenteralroutes, or by topical or transdermic routes.

The therapeutic dose varies, depending on the patient's age and weightas well as on the type of disease, from 0.1 to 100 mg/kg/day, preferablyfrom 1 to 30 mg/kg/day, over variable periods depending on the disease,in any case for at least 30 days.

The pharmaceutical compositions are inclusive of all formulationscontaining pharmaceutically acceptable excipients, that are suitable forthe administration of the claimed active ingredients in the forms bestsuited to the disease to be treated and, in any case, rendering theactive ingredients as bioavailable as possible. In particular,injectable solutions for general intravenous, subcutaneous andintramuscular administration and solutions for ophthalmic treatment areto be envisaged. As concerns the formulations per os granular powders,tablets, pills and capsules are the preferred ones.

EXAMPLE 1 Tablets

Every tablet contains:

    ______________________________________    N-palmitoylethanolamide                       300         mg    cellulose          40          mg    lactose            120         mg    sodium carboxymethylamide                       25          mg    precipitated silica                       20          mg    polyvinylpyrrolidone                       16.66       mg    maize starch       13.00       mg    Tween 20           7.14        mg    magnesium stearate 6.20        mg    ______________________________________

EXAMPLE 2 Vials for Injection

Every vial contains:

    ______________________________________    lyophilized N,N'-bis-                         10         mg    (2-hydroxyethyl)-nonandiamide    to be taken up in buffer solution:    dibasic sodium phosphate 12 H.sub.2 O                         6          mg    monobasic sodium phosphate 2 H.sub.2 O                         6          mg    sodium chloride      16         mg    Water for injectable formulations                         q.s. to 2 ml    ______________________________________

EXAMPLE 3 Gelatin Capsules

Every capsule contains:

    ______________________________________    N-arachidonylethanolamide                      50           mg    O.P. peanut oil   100          mg    O.P. gelatin      52           mg    O.P. glycerin     16           mg    erythrosine (E127)                      0.1          mg    ______________________________________

EXAMPLE 4 Gel for Ophtalmic Use

Every tube contains 5 g of product having the following composition:

    ______________________________________    w/w    N,N'-bis-(2-hydroxyethyl)-nonandiamide                          4          g    (as per Example 9)    benzalconium chloride 0.008      g    carbopol 940          3.5        g    disodium edetate      0.01       g    hydrochloric acid/sodium hydroxyde                          q.s. to pH 5    purified water        q.s. to 100 g    ______________________________________

In conclusion, the compounds of the present invention, when adequatelyformulated, may be conveniently used in human and animal therapy for thetreatment of diseases in which acute or chronic neurological damage isdirectly or indirectly due to excitotoxic phenomena, such as caused byGlu or related toxins. Said diseases are e.g.: hypoxic and/or ischemiccerebral insults, hypoxic and/or ischemic acute or recurrent andtransient attacks, such as TIA; brain or spinal cord injuries;neurodegenerative disorders of unknown etiology (Alzheimer's andParkinson's diseases, Huntington's Chorea, amyotrophic lateralsclerosis), or derived from neuronal disturbances following epilepsy andsevere hypoglycemic states; neurological complications whose primarycause is an infection (e.g. HIV); neurological complications followinghypoxic states caused by cardiovascular surgery or heart failure;diseases pertaining to the retina and in general to the visual system,as well as pertaining to other cranial nerves, e.g. nerves afferent tothe auditory system, and neurological diseases following excitotoxicdamage due to toxic agents e.g. neurolathyrism.

We claim:
 1. A method for treating or preventing neurotoxicity inducedby excitatory amino acids in a subject, comprising administering to thesubject an effective amount of the compoundN,N'-bis-(2-hydroxyethyl)-nonandiamide.
 2. The method according to claim1, wherein said neurotoxicity is associated with a disease selected fromthe group consisting of hypoxia-ischemia, stroke, brain and spinal cordinjuries, epilepsy, transient ischemic attacks, neurolathyrism,amyotrophic lateral sclerosis, Huntington's Chorea, Alzheimer's disease,primary dementia, dementia associated with viral infections, andanoxia-ischemic diseases of the retina.
 3. The method according to claim1, wherein said compound is orally administered in the form of agranular powder, tablet, pill or capsule.
 4. The method according toclaim 1, wherein said compound is administered intravenously,subcutaneously or intramuscularly.
 5. The method according to claim 1,wherein said compound is administered for ophthalmic use in the form ofdrops or ointments.
 6. The method according to claim 1, wherein saidcompound is administered topically or transdermally.
 7. The methodaccording to claim 1, wherein said compound is administered at a dosagein the range of 0.1 to 100 mg/kg/day for at least 30 days.
 8. The methodaccording to claim 7, wherein said dosage ranges from 1 to 30 mg/kg/day.